UNIVERSIDADE FEDERAL DO RIO GRANDE

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UNIVERSIDADE FEDERAL DO RIO GRANDE – FURG INSTITUTO DE CIÊNCIAS BIOLÓGICAS – ICB PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS FISIOLÓGICAS - FISIOLOGIA ANIMAL COMPARADA

Efeitos bioquímicos do herbicida Roundup no poliqueta estuarino Laeonereis acuta (Polychaeta, Nereididae)

Biólogo Fábio de Melo Tarouco

Dissertação apresentada como parte dos requisitos para obtenção do título de Mestre no Programa de Pós-Graduação em Ciências Fisiológicas - Fisiologia Animal Comparada, da Universidade Federal do Rio Grande - FURG, sob orientação do Prof. Dr. Carlos Eduardo da Rosa e Co-orientação do Dr. Marcio Alberto Geihs.

Rio Grande, Setembro de 2015.

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2 Agradecimentos

Em primeiro lugar agradeço à minha mãe pelo carinho e apoio que foram muito importante em todas as etapas dessa caminhada.

Às minhas irmãs Luciana e Selma pelo incentivo ao longo desses mais de dois anos e por acreditar que tudo isso seria possível.

À minha esposa e em especial aos meus amados filhos que são uma fonte inspiradora, me impulsionando em cada escolha que faço. Quem sabe toda essa caminhada não sirva de exemplo a ser seguido por eles? Pois embora todas as dificuldades impostas ao longo da vida, é sempre possível seguir e completar os objetivos traçados. Obrigado por existirem e amo incondicionalmente cada um de vocês.

Aos meus colegas, Amanda, Robson e Filipe que se mostraram além de colegas, verdadeiros amigos, fazendo parte dessa jornada, formando uma equipe nota 10.

Obrigado pelos risos, pela ajuda nas atividades de laboratório, mas principalmente pela atenção e pelo carinho que pude sentir durante esse tempo.

Aos amigos e colegas como a Fernanda Lopes, Fernanda Figueira, Lais, Regina e Marcelo pela companhia, pelos cafezinhos, pelas discussões polêmicas.

Não poderia deixar de agradecer ao meu orientador Nino, por ter aceitado fazer parte de mais esse desafio, por toda a dedicação que foram fundamentais no desenvolvimento desse trabalho. Saiba que te considero além de um profissional uma pessoa exemplar.

Obrigado por me acompanhar em toda essa caminhada.

Ao meu co-orientador Marcio, por toda a atenção, pelas trocas de protocolos, pelo acompanhamento durante o desenvolvimento dessa dissertação e pela ajuda no laboratório.

Por fim gostaria de agradecer a FURG, ao Instituto de Ciências Biológicas – ICB e ao Programa de Pós-Graduação em Ciências Fisiológicas – Fisiologia Animal Comparada por toda a estrutura física e humana, que foram importantes para o desenvolvimento do trabalho.

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3 Sumário

Resumo Geral...4

Introdução geral...5

Objetivo geral...14

Objetivos específicos...14

Manuscrito...15

Abstract...17

1. Introduction...18

2. Material and Methods...20

2.1. Animal collection and maintenance ...20

2.2. Experimental Procedures...20

2.3. Oxygen Consumption...21

2.4. Biochemical Analysis...21

2.4.1. Reactive Oxygen Species (ROS) determination and Antioxidant Capacity Against Peroxylradicals (ACAP) ...22

2.4.2. Antioxidant enzymes and GSH content ... 23

2.5. Lipid Peroxidation ...24

2.6. Cholinesterase activity ...25

2.7. Statistical analysis...25

3. Results...26

4. Discussion...31

5. Conclusion...37

6. Acknowledgements...37

Cited References...38

Discussão geral...46

Bibliografia geral...50

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4 Resumo Geral:

O Roundup é um herbicida que utiliza como princípio ativo o glifosato e é empregado no controle de ervas daninhas. Resíduos destas substâncias podem contaminar corpos d'água e causar danos à vida aquática. O objetivo foi avaliar a toxicidade do Roundup no poliqueta estuarino Laeonereis acuta, verificando a concentração que causa 50% de mortalidade e os efeitos relacionados com o balanço oxidativo e alterações na atividade das enzimas acetilcolinesterase (AChE) e propionilcolinesterase (PChE). Para os testes de toxicidade, os animais foram expostos por 96 horas em concentrações em ordem crescentes de Roundup, que variaram entre 0 (zero) L e 21,12 mg/L . Determinou-se que a CL₅₀ 96h é de 8,19 mg/L. Com base neste teste foram escolhidas duas concentração de Roundup (3,25 e 5,35 mg/L) para avaliar os demais parâmetros nos tempos 24 e 96h. Não foram observadas alterações no consumo de O2, porém houve uma redução nos níveis de espécies ativas de oxigênio (EAO) na região posterior nos dois tempos analisados. Verificou-se também que na maior concentração houve uma redução de ACAP nas três regiões corporais de L. acuta no tempo de 24h sendo e que após 96h de exposição a ACAP foi semelhante ao grupo controle em todos as regiões.

Não foram observadas alterações nas enzimas do sistema de defesas antioxidantes bem como nos níveis de GSH. Considerando-se a peroxidação lipídica, observou-se uma redução no tempo de 24h em todas as regiões analisadas. Já no tempo 96h houve uma redução na maior concentração testada na região anterior e um aumento na região média nos animais da menor concentração empregada. Uma redução na atividade da AChE e PChE foi observada após 96h de exposição à ambas concentrações na fração de membrana. Podemos concluir que o Roundup além de causar alterações no sistema colinérgico causa uma redução nos níveis de EAO e consequentemente da capacidade antioxidante, provavelmente devido a alterações no metabolismo intermediário.

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5 Introdução geral:

Segundo o último censo realizado no Brasil, entre os anos 1872 e 2010 a população brasileira teve um crescimento de quase 20 vezes, ultrapassando 190 milhões de habitantes (IBGE, 2010). Como consequência desse crescimento populacional, houve um aumento na demanda de alimentos. Para suprir essa demanda, se faz necessário o uso de diferentes técnicas visando melhorar a eficiência na produção agrícola. Entre as técnicas empregadas está o uso de agrotóxicos. Estes podem ser definidos como os produtos químicos e agentes físicos ou biológicos, destinados ao uso nos setores de produção, no armazenamento e beneficiamento de produtos agrícolas, nas pastagens, na proteção de florestas, nativas ou implantadas, e de outros ecossistemas e também de ambientes urbanos, hídricos e industriais, cuja finalidade seja alterar a composição da flora ou da fauna, a fim de preservá-las da ação danosa de seres vivos considerados nocivos (Lei Federal n°7.802/89).

Em 2008 o Brasil assumiu o primeiro lugar no consumo de agrotóxicos ultrapassando os EUA, se tornando assim o maior consumidor de agrotóxicos do mundo (ANVISA, 2010). Entre os agrotóxicos, está a classe dos herbicidas, substâncias químicas que reduzem ou eliminam plantas daninhas, que competem por água e nutrientes, ocasionando perdas nos cultivos (IBAMA, 2010). O controle das ervas daninhas é de fundamental importância para a obtenção de produtividade em qualquer exploração agrícola, sendo tão antiga quanto a agricultura (EMBRAPA, 2003).

No Brasil, o crescente aumento no consumo de agrotóxicos, ocorre devido o aumento das áreas onde é praticado o plantio, sendo que os herbicidas lideram a lista de agrotóxicos mais consumidos, superando 127 mil toneladas em 2009 (IBAMA, 2010).

Entre os herbicidas mais utilizados, estão os que utilizam o glifosato como princípio ativo, o qual ultrapassou 90 mil toneladas em 2009, representando 76% do total de

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6 herbicidas utilizados no Brasil (IBAMA, 2010). Seu uso não está restrito ao agrícola, sendo empregado na silvicultura, horticultura e ainda nos espaços urbanos para o controle de ervas daninhas (USEPA, 2011).

O glifosato (figura 1) é um herbicida não seletivo que tem como alvo a enzima 5-enolpiruvilchiquimato-3 fosfatosintase (EPSPS). Esta enzima é chave na via do chiquimato, envolvida na biossíntese de aminoácidos aromáticos. Porém, esta via só está presente em plantas e micro-organismos e ausente em animais (Giesy et al., 2000).

Este herbicida está classificado na classe III, que leva em consideração o potencial de periculosidade ambiental, (IBAMA, 2009).

Figura 1 - Estrutura química do glifosato. Fonte: Dzygiel e Wieczorec, 2000.

Uma das preocupações pelo crescente uso de herbicidas é a contaminação dos corpos d'água, que pode ocorrer de diferentes formas como, por exemplo, devido o processo de lixiviação, erosão ou devido à aplicação de forma inadequada do produto.

Segundo a resolução CONAMA nº 357, de 17 de março de 2005, o limite máximo de glifosato permitido em águas doces de classe 1, destinadas ao abastecimento para consumo humano, à preservação do equilíbrio natural das comunidades aquáticas e à preservação dos ambientes aquáticos em unidades de conservação de proteção e preservação da fauna e flora aquática é de 65 µg/L. No entanto já foram encontrados níveis ambientais superiores ao estabelecido pela resolução CONAMA. No trabalho de

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7 Silva et al. (2003), na qual avaliaram a presença de glifosato 30 e 60 dias após a aplicação do herbicida, nas águas superficiais da microbacia hidrográfica arroio Passo do Pilão, localizado no Sul do Estado do Rio Grande do Sul foram encontrados concentrações em diferentes pontos, que variaram de 20 µg/L a 30 µg/L, até concentrações superiores a 100 µg/L, em pontos próximos a locais onde ocorre o cultivo de milho.

No mercado atualmente existem diferentes formulações comerciais, que utilizam o glifosato como princípio ativo. Entre essas formulações podemos destacar o Roundup, que além de princípio ativo, apresenta entre seus compostos, ingredientes, ditos inertes além de água e surfactantes. O surfactante mais utilizado nas formulações à base de glifosato é o polioxietilenoamino (POEA), utilizado para facilitar a absorção do glifosato através da cutícula das plantas, sendo que a quantidade do surfactante utilizado pode chegar em torno de 15% da formulação (Giesy et al., 2000). O tempo estimado para meia vida do glifosato em ambientes aquáticos fica em torno de 7 a 14 dias (Giesy et al., 2000).

Embora o glifosato não tenha um alvo específico em animais, já foi demonstrado que tanto puro quanto nas suas formulações comerciais podem afetar vários parâmetros como, por exemplo, efeitos em nível de metabolismo energético (Peixoto, 2005), dano de DNA (Nwani et al., 2013) e no sistema reprodutivo (Lopes et al., 2014). Além desses efeitos, já foi relatado que a exposição a herbicidas à base de glifosato, pode causar estresse oxidativo.

O estresse oxidativo pode ser definido como resultado de um desequilíbrio no balanço entre pró-oxidantes e antioxidantes, em favor dos pró-oxidantes (Sies, 1991).

No entanto Jones (2006) propôs uma nova definição levando em consideração a relação de que todos os sistemas biológicos contêm elementos redox, e esses elementos

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8 possuem um importante papel na sinalização celular e assim na regulação fisiológica.

Sendo assim, além do conceito clássico, o estresse oxidativo, é também definido como, qualquer perturbação da função sinalizadora que possuem essas espécies ativas de oxigênio (EAO).

As EAO são produzidas de forma natural durante o processo de respiração celular na cadeia transportadora de elétrons, na qual durante a redução do oxigênio a água, são gerando espécies intermediárias de oxigênio, como o radical ânion superóxido (O2^.-), o radical hidroxila (HO^.), e o peróxido de hidrogênio (H₂O₂) (Figura 2). Em situações normais, estima-se que cerca de 0,1 % do oxigênio consumido em mamíferos, possa gerar essas EAO (Fridovich, 2004). As EAO podem reagir com biomoléculas como proteínas, fosfolipídios e DNA, causando danos ao funcionamento celular (Meneghini, 1987).

Figura 2 - Redução do O2 a água com a formação de espécies ativas de oxigênio.

Fonte: Adaptado de Lushchak 2011.

Para neutralizar ou reduzir os efeitos indesejados das EAO os organismos possuem um sistema de defesas antioxidantes (SDA) (Vercesi, 2003). Essas defesas podem ser enzimáticas (figura 3) representadas, por exemplo, por enzimas como a superóxido dismutase (SOD), que acelera a conversão do ânion superóxido em peróxido de hidrogênio e oxigênio, impedindo o efeito tóxico direto deste radical; a catalase (CAT) e glutationa peroxidase (GPx) que participam da metabolização do peróxido de

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9 hidrogênio; a glutationa S-transferase (GST) que catalisa a conjugação de glutationa reduzida (GSH) com xenobióticos como, por exemplo produtos de dano oxidativo, como os lipídeos peroxidados, e a enzima glutamato-cisteína ligase (GCL) que participa da biossíntese da GSH, desempenhando um importante papel na defesa antioxidante e no metabolismo de xenobióticos (Dickinson & Forman, 2002). Além do SDA enzimático, os organismos também contam com defesas não enzimáticas representadas, por exemplo, pelo ácido ascórbico, o tocoferol, o tripeptídeo glutationa reduzida (GSH) entre outras (Storey, 1996).

Figura 3 - Defesas antioxidantes enzimáticas, superóxido dismutase (SOD), catalase (CAT), Glutationa S-transferase (GST), glutationa peroxidase selênio dependente (GPx-Se), glutationa redutase (GR). Fonte: Adaptado da dissertação de Ferreira- Cravo, 2006.

No entanto em situações pró-oxidantes, pode ocorrer alterações em componentes do SDA, e com isso levar a um desbalanço no estado redox. Nesse contexto, já foi

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10 descrito que o glifosato pode afetar a atividade de enzimas envolvidas no sistema de defesa antioxidante (SDA), Contardo-Jara et al. (2009), verificaram aumento na atividade das enzimas SOD, CAT e GST no anelídeo Lumbriculus variegatus exposto por 96h em concentrações que variaram de 0,05 à 5mg/L tanto de glifosato quanto a formulação Roundup, indicando com isso que a exposição ao glifosato pode levar a uma situação de estresse oxidativo. Modesto & Martinez (2010a, 2010b) observaram no peixe Prochilodus lineatus exposto em concentrações de 1, 5 e 10 mg/L de Roundup, uma inibição no SDA, através da redução na atividade das enzimas SOD, CAT e GST.

Essa perturbação no SDA levou a um aumento nos níveis de lipídeos peroxidados.

Resultados semelhantes, foram encontrados no trabalho de Lushchak et al., (2009) na qual observaram alterações na atividade das enzimas SOD, CAT, GST e glutationa redutase (GR) no “goldfish” exposto a concentrações de Roundup que variaram de 2,5 a 20 mg/L de Roundup, reforçando assim a toxicidade do Roundup relacionado com balanço oxidativo.

Além de alterações no balanço oxidativo, já foi relatado que a exposição ao glifosato, embora este não seja um herbicida conhecido por afetar o sistema nervoso, (Amarante Junior et al., 2002), pode levar a alterações na atividade de colinesterases.

As colinesterases são importantes enzimas que possuem a função, de realizar a hidrólise de ésteres de colina (figura 4). Esses ésteres são importantes neurotransmissores que participam da regulação da transmissão do impulso nervoso na sinapse nervosa e na junção neuromuscular (Samadi et al., 2007). Nesse sentido Sandrini et al., (2013) observaram que a exposição ao glifosato in vitro pode inibir a atividade colinesterásica no mexilhão Perna perna e nos peixes Danio rerio e Jenynsia multidentata. Da mesma forma, Modesto & Martinez (2010a, 2010b) relataram uma redução na atividade da

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11 enzima acetilcolinesterase (AChE) no cérebro e no músculo do peixe Prochilodus lineatus exposto por 96h em concentrações de 1 e 5 mg/L de Roundup.

Figura 4 - Síntese, liberação e degradação da acetilcolina. Acetilcoenzima A (Acetil CoA), coenzima A (CoA), colina acetiltransferase (ChAT), acetilcolina (ACh), acetilcolinesterase (AChE). Fonte: Adaptado de Samad et. al., 2007.

Como mencionado anteriormente, ambientes aquáticos podem ser o destino final de alguns contaminantes. Dentre esses ambientes suscetíveis a contaminações de origem antrópica estão os estuários, regiões de transição fazendo a ligação entre rios, lagos e os mares ou oceanos, essas regiões podem receber contaminantes decorrentes de atividades industriais, portuárias e da agricultura (Saiz-Salinas e González-Oreja, 2000). Além de serem receptores desses contaminantes vindos do continente, também são considerados ambientes naturalmente estressados devido a grande variação nas características físico-

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12 químicas como, por exemplo, concentração de oxigênio dissolvido, salinidade, temperatura e pH (D’Incao et al., 1992; Elliott e Quintino, 2007). Sendo assim, os organismos que vivem nesse ambiente, estão constantemente expostos a estes estressores naturais bem como aos estressores de origem antrópica. Dada esta característica, possuem mecanismos que permitem neutralizar ou reduzir os possíveis danos relacionados às variações desses fatores abióticos, possibilitando assim a vida nesses ambientes.

Entre os organismos que vivem nas regiões estuarinas está o poliqueta Laeonereis acuta (Treadwell, 1923). Esta é uma espécie encontrada na costa atlântica da América do Sul (Omena e Amaral, 2001). Este animal vive em tocas construídas nos sedimentos destas regiões, destacando-se pela frequência de ocorrência tanto em locais poluídos quanto não poluídos (Ferreira-Cravo et al., 2007). Além disso, é considerado um importante elo nas teias alimentares tendo em vista ser um consumidor de detritos não-seletivo, além de ser item de alimentação de invertebrados, peixes e aves (Botto et al., 1998; Palomo e Iribarne, 2000; Palomo et al., 2004). Esta espécie tem sido utilizada como modelo experimental, frente à exposição a diferentes agentes pró-oxidantes de origem natural como o peróxido de hidrogênio (H2O2), na qual foi demonstrado que a exposição nas concentrações de 10µM e 50µM pode levar a uma perturbação no balanço oxidativo, causando alteração na atividade de enzimas antioxidantes como a SOD, CAT e GST bem como um aumento de LPO (Rosa et al., 2005). Nesse mesmo trabalho, Rosa et al., 2005 relataram para L. acuta, um gradiente corpóreo na atividade das enzimas CAT e SOD nesta espécie, sendo observado uma maior atividade dessas enzimas na região posterior, seguido pela região média e na região anterior na qual foi a verificada uma menor atividade, demonstrando com isso, um padrão de resposta diferente ao longo do corpo. Há também relatos dos efeitos de contaminantes de origem

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13 antrópica em L. acuta. Geracitano et al. (2002) verificaram que a exposição ao cobre nas concentrações de 250 µg/L e 500 µg/L por 48h (exposição aguda), causou um aumento de LPO. Além disso, Sandrini et al. (2008) observaram que a exposição a 100 µg/L de cádmio por 7 dias causa um aumento nos níveis de EAO e na atividade da GST além de redução na atividade da SOD. Resultados semelhantes relacionados ao estresse oxidativo foram observados por Ventura-Lima et al. (2011), os quais também relataram alterações em componentes do SDA, como a redução da GST em L. acuta exposto a 50 µg/L arsênico por 7 dias. Esses trabalhos mostram as respostas de L. acuta em nível do SDA frente à agentes pró-oxidantes de origem natural bem como de origem antrópica.

Levando em consideração o crescente aumento no uso dos herbicidas a base de glifosato e sabendo-se que embora este não apresente um alvo específico em animais, alguns estudos demonstram efeitos danosos à vida animal. Portanto, se fazem necessárias novos estudos visando elucidar os mecanismos de toxidade desse herbicida em outras espécies. Para tanto, no presente trabalho avaliamos os efeitos do herbicida Roundup, para o poliqueta estuarino L. acuta, relacionados com parâmetros do SDA bem como na atividade das enzimas AChE e PChE.

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14 Objetivo geral:

- Avaliar os efeitos do herbicida Roundup no balanço oxidativo bem como sua influência no sistema colinérgico no poliqueta Laeonereis acuta.

Objetivos específicos:

- Determinar a concentração de Roundup que causa 50% de mortalidade (CL₅₀) para L.

acuta.

- Avaliar os efeitos da exposição à concentrações subletais de Roundup relacionadas ao consumo de O2.

- Analisar o efeito do Roundup em concentrações subletais sobre a geração de espécies ativas de oxigênio, sistema de defesas antioxidantes e dano oxidativo nas diferentes regiões corporais de L. acuta.

- Avaliar in vivo os efeitos da exposição ao Roundup sobre a atividade colinesterásica de L. acuta.

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15 Manuscrito

Effects of the herbicide Roundup on oxidative balance and cholinesterase activity of the polychaeta Laeonereis acuta

( a ser submetido à revista Comparative Biochemistry and Physiology - Part C) Fator de Impacto 2,301

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16 Effects of the herbicide Roundup on oxidative balance and cholinesterase activity

of the polychaeta Laeonereis acuta

Fábio de Melo Taroucoâ, Filipe Guilherme Andrade de Godoi^b, Robson Rabelo Velasquesâ, Amanda da Silveira Guerreiroâ, Marcio Alberto Geihsâ, Carlos Eduardo da Rosaâ*.

aUniversidade Federal do Rio Grande - FURG, Av. Itália km 8, 96203-900, Rio Grande, RS, Brazil. Instituto de Ciências Biológicas, Programa de Pós-Graduação em Ciências Fisiológicas – Fisiologia Animal Comparada.

bUniversidade Federal do Rio Grande, Av. Itália km 8, 96203-900, Rio Grande, RS, Brazil.

e-mails:

[email protected] (Fábio de Melo Tarouco)

[email protected] (Filipe Guilherme Andrade de Godoi) [email protected] (Robson Rabelo Velasques) [email protected] (Amanda da Silveira Guerreiro) [email protected] (Marcio Alberto Geihs)

[email protected] (Carlos Eduardo da Rosa)

Correspondence author: Carlos Eduardo da Rosa ([email protected])

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17 Abstract:

Glyphosate based herbicides, such as Roundup, are widely employed in agriculture, silviculture and urban spaces to control the growth of weeds. However, these products contaminate river and estuarine systems, affecting aquatic life. The objective of the present study was to evaluate the effects of Roundup on the estuarine polychaeta Laeonereis acuta, considering oxidative balance as well as acetylcholnesterase (AChE) and propionilcholinesterase (PChE) activity. Animals were exposed to a range of Roundup concentrations in order to determine LC₅₀ at 96h, which was established as 8.19 mg/L. Based on this, the animals were exposed to two Roundup concentrations:

3.25 mg/L (NOEC) and 5.35 mg/L (LC10) for 24h and 96h. Oxygen consumption (VO2) was then determined and the animals were divided into three body regions (anterior, middle and posterior) for biochemical analysis. A significant reduction in ROS generation was observed in the posterior region of animals in both experimental periods, while antioxidant capacity was reduced in the posterior region of animals exposed for 24h. With respect to the antioxidant defense system, both GSH levels and enzymes (CAT, SOD, GST, GP_Xand GCL) were not altered after exposure. Lipid peroxidation was reduced in all analyzed body regions in both Roundup concentrations after 24h.

Animals exposed to the highest concentration of Roundup also presented a reduction in lipid peroxidation in the anterior region after 96h, while animals exposed to the lowest concentration presented a reduction in the middle region. A reduction in cholinesterase activity, including both AChE and PChE in animals exposed to both Roundup concentrations after 96h was observed. Overall results indicate that Roundup exposure presents toxicity to L. acuta, causing a disruption in ROS and ACAP levels as well as affects the cholinergic system of this invertebrate species.

Keywords: antioxidant defense, Roundup, invertebrate, Nereididae.

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18 1. Introduction:

Agrichemicals are one of the most widespread contaminants observed in the aquatic environment (Silva et al., 2003). Glyphosate based herbicides such as Roundup are the most employed in agriculture, comprising up to 40% of pesticides commercialized in Brazil (Carneiro et al., 2015). Besides its use in agriculture, Roundup can be used in silviculture, horticulture and in various domestic cases (USEPA, 2011). Its primary action is as a non-selective herbicide employed in weed control by the inhibition of the enzyme 5-enolpyrivilshikimate-3 phosphate synthase (EPSPS), which is a key enzyme in the shikimate pathway and responsible for the biosynthesis of aromatic amino acids in plants and microorganisms (Giesy et al., 2000).

Although animals do not possess the physiological targets for specific glyphosate toxicity, it has been demonstrated that both the active ingredient and its commercial formulations affect physiological and biochemical parameters in animals.

These effects include: the inhibition of cell respiration due to a reduction in the mitochondrial membrane potential and the activity of ATP synthase in isolated mitochondrion of rats (Peixoto, 2005), a reduction in acetylcholinesterase activity in mollusks and fish (Sandrini et al. 2013) and reproduction impairment in fish (Lopes et al., 2014). Besides these effects, it has been proposed that glyphosate exposure may cause alterations in enzymes of the antioxidant defense system in annelids (Contardo–

Jara et al., 2009). Such alterations to enzymes responsible for maintaining the cell redox state would lead to an imbalance between prooxidants and antioxidants in favor of the former. This then leads to a disturbance in cell redox signaling and oxidative damage, a situation called oxidative stress (Sies,1991; Jones, 2006). Based on this, Roundup exposure may lead to a similar situation in aquatic organisms such as the fish species Carassius auratus and Prochilodus lineatus leading to the alteration of antioxidant

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19 enzyme activities such as superoxide dismutase (SOD), catalase (CAT), glutathione S- transferase (GST) and glutathione reductase (GR) (Lushchak et al., 2009; Modesto e Martinez, 2010b).

Aquatic environments represent the final destination for a variety of anthropogenically based contaminants, including those introduced by industrial activity and agriculture (Saiz-Salinas e González-Oreja, 2000). Among these aquatic recipients, estuaries are considered highly stressful environments due to their great variability in salinity, dissolved oxygen, temperature and pH (D’Incao et al., 1992; Elliott e Quintino, 2007), which may be exacerbated by contaminants. Consequently, organisms that live in such environment are exposed to both natural and anthropogenic stimulus. These organisms consequently possess adaptations in order to neutralize or reduce damage caused by such variation in abiotic factors.

Polychaetes are among the organisms that inhabit estuarine environments, and Laeonereis acuta represents one of the primary Brazilian species (Treadwell, 1923).

This benthonic species occurs in the Atlantic shores of South America (Omena and Amaral, 2001), living in burrows constructed within sediment. Such organisms are important elements in the food chain since they are non-selective filter-feeders and represent a food source for invertebrates, fish and birds (Botto et al., 1998; Palomo e Iribarne, 2000; Palomo et al., 2004). This polychaete species has been employed as an experimental model for assessing the effects of pro-oxidant agents of both natural and anthropogenic sources, such as hydrogen peroxide (Rosa et al., 2005), copper (Geracitano et al., 2002), cadmium (Sandrini et al., 2008) and arsenic (Ventura-Lima et al., 2011).

Since polychaetes inhabit sediments from estuarine environments that represent a common recipient of pollutants, the objective of the present study was to evaluate

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20 toxic effects of Roundup in L. acuta with respect to mortality, oxidative balance and cholinesterase enzyme activity.

2. Material and Methods:

2.1. Animal collection and maintenance

Laeonereis acuta were collected in the Patos Lagoon Estuary in Rio Grande, Southern Brazil and transferred in plastic bags containing cold water from the collection site. The license for collection was provided by the Chico Mendes Institute for Biodiversity Conservation – Brazil (ICM-Bio / license number 40868-1). Animals were acclimated for 7 days under lab conditions in plastic boxes containing sediment and saline water (10ppm), temperature 20± 2°C, pH 8.0 and photoperiod 12L:12D. Animals received commercial fish food every 2 days, and the water was renewed every second day.

2.2. Experimental Procedures:

Since Roundup Original is a complex mixture composed of both glyphosate (360 g/L) and inert ingredients, Roundup concentrations in the present study were based on the relative glyphosate equivalents. In order to define sub-lethal Roundup concentrations to L. acuta for further physiological and biochemical analyses, polychaetes were exposed to Roundup concentrations from 0.065 mg/L (representing the maximum commercial concentration with respect to the preservation of flora and fauna according to Brazilian legislation - CONAMA 357) to 65 mg/L. 10 animals per treatment in individual flasks (100mL) were exposed to herbicide for 96h. Initially, five test concentrations (in a geometric series) and a control group were used. The test was then repeated employing three concentrations (in a geometric series) ranging from the lowest concentration that resulted in 100% mortality and the highest concentration that did not cause any mortality. In all sets of experiments, mortality was verified daily and

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21 the experimental water was renewed every 48h (semi-static system). Animals were exposed without sediment and water conditions and were maintained during the acclimation period. Results were analyzed by the Probit method (EPA Probit Analysis Program - version 1.5); the CL50 and CL10 96h were determined as well as the non- observed effect concentration (NOEC). For the posterior tests, the animals were exposed to Roundup 3.25 and 5.35 mg/l, the NOEC and CL₁₀respectively.

2.3. Oxygen Consumption:

Animal oxygen consumption was determined according to the methodology of Nithart et al. (1999). Animals (n=10) were transferred to glass flasks containing water (10mL) at the same characteristics of salinity, pH and temperature as the exposure period. The initial oxygen concentration was recorded with an oxymeter (DO-5519, Lutron Eletrônica – CO), after which the flasks were sealed for 20 minutes. Then, the flasks were opened and the final oxygen concentration was determined. The animals were weighed and flask volumes were verified. The oxygen consumption was expressed as mg O2/mL/mg wet weight/hour.

2.4. Biochemical Analysis:

After the exposure periods, each animal was divided into anterior region (20 initial segments), middle region (next 20 segments) and posterior region (final segments). Such division was based on previous studies that determined differences in the antioxidant responses in distinct regions of the animal body (Rosa et al., 2005). This division was employed for all the biochemical determinations, except for cholinesterase activity, in which the anterior region was the only one employed.

2.4.1. Reactive Oxygen Species (ROS) determination and Antioxidant Capacity Against Peroxyradicals (ACAP):

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22 In order to determine ROS generation and ACAP, polychaeta were exposed for 24h and 96h, and immediately sacrificed in ice and divided as described above. Pools from each body region of two animals (n=5). Pools were homogenized (1:5 W/V) in cold buffer (100 mM Tris-HCl, 2 mM EDTA, 5 mM MgCl2). Samples were then centrifuged for 20 minutes at 20,000 x g and 4°C. The supernatant was taken for subsequent analyses. The protein content from samples was determined according to the biuret method with a commercial kit employing bovine serum albumin as standard (Doles Reagents). Consequently, all the samples were diluted with the homogenization buffer to a final protein concentration of 2 mg/mL.

In order to quantify ROS, sample aliquots were added to a microplate with cold buffer (HEPES 30mM, KCl 200mM and MgCl₂ 1mM). After, the 2',7'- dichlorofluorescein diacetate (H2DCF-DA, 16 µM, Molecular Probes) was added.

Dichlorofluorescein diacetate is hydrolyzed by esterases to a non-fluorescent form, DCFH, which in turn is oxidized by ROS. This oxidation generates a fluorochrome detectable in a microplate reader (Victor 2, Perkin Elmer) at excitation and emission wavelengths of 488 nm and 525 nm, respectively (LeBel et al., 1992).

ACAP determination followed the methodology of Amado et al. (2009). This procedure is based on the thermal decomposition (at 37°C) of 2,2'-azobis (2 methylpropionamidine) dihydrochloride (ABAP 20mM; Sig-Aldrich), which generates peroxyradicals. These molecules react with H2DCF-DA, subsequently forming a detectable fluorochrome which was analyzed using the methods of the above description. The antioxidants present in the sample react with the peroxyradicals, consequently reducing the fluorescence generation. ACAP was expressed as relative fluorescence area, and was determined as following:

ACAP = 1/(fluorescence area with ABAP - without ABAP)/area without ABAP

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23 2.4.2. Antioxidant enzymes and GSH content:

A total of 3 animals for each body region (n=5) were used for enzymatic determination. After the exposure period, animals were dissected on ice and samples were homogenized (1:5 W/V) in ice buffer (20 mM Tris base, 1mM EDTA, 1 mM dithiothreitol, 500 mM sacarose, 150 mM KCl e 0.1 mM PMSF, pH 7.6). An identical procedure was employed for glutamate cysteine ligase activity determination (ɣGCL) and for reduced glutathione (GSH) content analysis; however a different buffer was used (100 mM tris-HCl, 2 mM EDTA, 5 mM MgCl₂). Samples were then centrifuged for 20 minutes at 9,000 x g and 4°C. Protein concentration was determined as previously described and the supernatant was frozen for subsequent analysis (-80°C).

Catalase activity (CAT) was determined following the protocol described by Beutler (1975). This method is based on the degradation of hydrogen peroxide (50mM), which is spectrophotometrically monitored (240nm). Results were expressed in CAT units, which are defined as the amount of enzyme needed to hydrolyze 1 µmol H2O2 per minute and per protein (mg) at 30°C and pH 8.0.

Superoxide dismutase activity (SOD) was analyzed using spectrophotometric monitoring of adrenochrome formation at 480nm. Adrenochrome formation occurs via the oxidation of epinephrine by the superoxide anion present in the buffer. Results were expressed as SOD units, with one unit representing the amount of enzyme needed to inhibit 50% of epinephrine oxidation (Misra and Fridovich, 1972).

Glutathione S-transferase (GST) activity was determined according to Habig and Jakoby (1981). This assay is based on the product formation by the conjugation of glutathione with 1-chloro, 2,4-dinitrobenzene (CDNB), which is determined by spectrophotometry (340nm). Results were expressed in GST units, which are defined

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24 as the amount of enzyme needed to generate 1 µmol of product per minute and per mg of protein at 25°C and pH 7.00.

Glutathione peroxidase (GPx) activity was determined according to Arun and Subramanian (1998). This method determines NADPH (0.12mM) oxidation in a reaction media containing phosphate buffer (100mM; pH 7.5), glutathione reductase (0.1U/ml), GSH (2mM), H₂O₂ (2mM) and sodium azide (1mM) via spectrophotometry (340nm). Results were expressed as GPx units, which are defined as the amount of enzyme necessary to oxidize 1 ɲmol of NADPH per minute and per mg of protein at 30°C.

ɣGCL and GSH content were analyzed according to White et al. (2003). This method is based on the reaction of dicarboxialdehyde (NDA) with GSH or the ɣ- glutamilcysteine residues to form highly fluorescent cyclic products. Fluorescence intensity was measured at 472nm (excitation) and 528nm (emission) in a microplate fluorescence reader (Victor 2, Perkin Elmer). ɣ-GCL activity was expressed as GSH ɲmoles per hour and per mg of protein and GSH content was expressed as mg of GSH per mg of protein.

2.5. Lipid Peroxidation:

Lipid peroxidation was determined according to the protocol from Hermes-Lima et al. (1995). Pools from two animals for each body region were homogenized in cold methanol (100%) at a ratio of 1:9 (W/V). Thereafter, homogenates were centrifuged at 1,000 x g for 10 minutes at 4 °C. The resulting supernatant was taken for the quantification of lipid hydroperoxides (LPO), via the FOX method in a microplate reader at 550nm. Cumene hydroperoxide (CHP) was employed as a standard and the results were expressed as CHP equivalents per wet weight.

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25 2.6. Cholinesterase activity:

In order to determine cholinesterase activity, animal tissue samples were prepared according to the methodology of Sandrini et al. (2013). Samples consisted of pools (n=6) from 4 anterior regions, homogenized in 200 µL of cold phosphate buffer (100mM) mixed with glycerol 20%. Homogenates were centrifuged at 9,000 x g for 30 minutes at 30°C; the resulting supernatant was employed as the soluble fraction (SF).

Pellets were re-suspended in homogenization buffer mixed with Triton X-100 (0.5%) and incubated for 30 minutes at room temperature. Following incubation, re-suspended pellets were centrifuged with the same conditions described above. The supernatant from this centrifugation was employed as the enzyme source for the membrane fraction (MF).

Enzyme activity was determined via the method described by Elman et al.

(1961). DTNB (5,5-dithiobis-2-nitrobenzoic acid – Sigma-Aldrich) and acetylthiocholine (7.5 mM) or propionylthiocholine (4mM) were used. Enzyme activity was analyzed using a microplate reader (412nm). Results were expressed as ɳmoles/mg of protein/min. Protein concentrations from supernatants were measured by the biuret method as described before.

2.7. Statistical analysis:

Statistical analyses included the one-way Anova test (assumptions of normality and homoscedasticity were validated) followed by the Newman-Keuls post hoc test (p<0.05). Under circumstances in which the assumptions of Anova were not met, the non-parametric test of Kruskal-Wallis Anova was used. The values were presented as means ± standard error.

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26 3. Results:

Based on toxicological tests (Fig. 1) in which animals were exposed to a wide range of Roundup concentrations for 96h, concentrations that caused 10% and 50%

mortality in polychaetes were determined as 5.35 mg/L and 8.19 (C.I. 6.69 -9.58 mg/L), respectively.

Figure 1- Percent mortality of the polychaeta L. acuta exposed to different Roundup concentrations for 96h (Results from the final test described in the material and methods section).

It is important to note that during the following experiments no significant mortality was observed. Concerning oxygen consumption (Fig. 2A and 2B), no alteration in both Roundup concentrations and both experimental periods relative to the control group animals (p>0.05) was observed.

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27 Figure 2- Oxygen consumption of L. acuta exposed to two Roundup concentrations (3.25 and 5.35 mg/L) for 24h (A) and 96h (B). Data are expressed as means ± standard error.

A reduction in ROS production in the posterior regions of animals exposed to both Roundup concentrations after 24h comparing to control group animals was observed (Fig. 3A). A reduction of 1.7 times was observed for the lowest concentrations in comparison with control group animals (p<0.05), while a reduction of 2.6 times was observed for the highest concentrations. After 96h a significant alteration was observed between the posterior body region of worms exposed to Roundup (p<0.05) such that ROS generation was 2.7 times lower in animals exposed to the higher concentration when compared to the lowest concentration (Fig. 3B). No significant alteration was observed between animals exposed to Roundup and control group animals in this exposure period (p>0.05). Other body regions did not present any significant alteration in ROS generation for both periods analyzed (p>0.05).

A significant reduction in ACAP levels was observed (p<0.05) in the three body regions of animals exposed to the highest concentration of Roundup when compared with control group animals after 24h (Fig. 3C). Alterations of 1.8, 1.6 and 2.1 times in the anterior, middle and posterior region respectively were observed. On the other hand, no alteration in ACAP levels was observed in both experimental groups after exposure for 96h (p>0.05); ACAP levels returned to values similar to that observed in control group animals (Fig. 3D).

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28 Figure 3- Reactive Oxygen Species (ROS) in the different body regions of L. acuta exposed to Roundup for 24h (A); 96h (B); Antioxidant Capacity Against Peroxylradicals after 24h (C); and 96h (D). Data are expressed as means ±standard error. Different letters represent significant differences between treatments at the same body region and experimental period (p<0.05).

No significant difference in the activity of CAT, GST and GPx enzymes was observed in both Roundup concentrations used in both exposure periods (Table 1). SOD enzyme activity was 1.7 times lower in the middle region from animals exposed to the higher Roundup concentration when compared to the lowest concentration after 24h (p<0.05). Contrarily, SOD activity after 96h remained similar between all experimental groups (p>0.05). No significant difference was observed between groups for GCL activity and GSH levels.

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29

Table 1. Activity of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione S-transferase (GST), glutamate cysteine ligase (GCL) and GSH levels in different regions of L. acuta exposed to Roundup. Data were expressed as means ± standard errors.

24hours 96hours

Roundup (mg/L)

0 3.25 5.35 0 3.25 5.35

CAT

Anterior 1.00±0.21 0.80±0.05 0.93±0.19 0.77±0.14 1.04±0.26 0.68±0.18 Middle 1.09±0.07 1.16±0.22 0.93±0.08 0.58±0.11 0.68±0.09 1.12±0.36 Posterior 1.59±0.32 1.08±0.12 1.13±0.05 1.05±0.20 1.33±0.34 1.41±0.51

SOD

Anterior 0.65±0.05 0.49±0.05 0.50±0.03 0.47±0.06 0.59±0.05 0.59±0.02 Middle 0.51±0.04^a,b 0.65±0.06^a 0.40±0.02^b 0.53±0.05 0.65±0.05 0.62±0.06 Posterior 0.66±0.05 0.52±0.02 0.61±0.02 0.74±0.11 0.72±0.10 0.75±0.05

GPx

GST

GCL

GSH

Values are expressed as: CAT (U CAT), SOD (U SOD), GPx (U GPx), GST (U GST), GCL (ɲM GSH), GSH (ɲmoles)Different letters represent statistical differences within the same region in the different treatments.

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30 Significant differences in lipid peroxidation were observed, with results indicating reductions of 7.9, 6.7 and 4.8 times in the anterior, middle and posterior region, respectively, in animals exposed to the higher Roundup concentration when compared with the control group animals (p<0.05). Similarly, LPO levels in the middle region of animals exposed to the lowest herbicide concentration were reduced by 2.7 times when compared with control group animals. LPO levels were reduced by 5.0 times for groups exposed to the highest concentration for 96h compared to control groups (p<0.05). Animals exposed to the lowest herbicide concentration presented an increase of 3.5 times in LPO levels in the middle region. Lipid peroxidation in the posterior region was similar to control group animals in both Roundup concentrations (p>0.05).

Figure 4- Lipid hydroperoxide levels in the anterior, middle and posterior region in the distinct body regions from Laeonereis acuta exposed to Roundup for 24h (A) and 96h (B). Data is represented as means ± standard errors. Different letters represent significant differences between treatments at the same body region and experimental period (p<0.05).

No significant alteration was observed for cholinesterase activity (p>0.05) in animals exposed to both Roundup concentrations in both SF and MF (Fig 5A and 5C, respectively). However, after 96h of Roundup exposure a significant reduction of 1.5 times (p<0.05) in AChE activity in animals exposed to both Roundup concentration in the MF (Fig. 5B) was observed. Similar results were observed to PChE activity,

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31 wherein a significant reduction (p<0.05) of 1.4 times was observed in the same fraction (MF) compared to the respective control group (Fig. 5D). SF, AChE and PChE activity was similar between all experimental groups (p>0.05).

Figure 5- Acetylcholinesterase activity (AChE) (24h-A and 96h-B) and propionylcholinesterase activity (PChE) (24h-C and 96h-D) from Laeonereis acuta exposed to Roundup. Data is represented as means ± standard errors. Different letters represent significant differences between treatments at the same body region and experimental period (p<0.05).

4. Discussion:

The herbicide Roundup does not represent a substance that may specifically target animals, due to the absence of the EPSPS enzyme; however, studies have reported its toxicity to several organisms. In this sense, Mensah et al.(2013) described the LC₅₀ for different vertebrate and invertebrate species: 12.24 mg/L for the insect Tanytarsus flumineus, 4.30 mg/L to the mollusk Burnupia stenochorias, 2.84 mg/L and 0.657 mg/L for the crustacean species Caridina nilotica and Daphnia pulex and 3.25 mg/L for the fish Oreochromis mossambicus. In addition, Yadav et al. (2013) reported

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32 the Roundup concentration of 3.39 mg/L as the LC₅₀ for the amphibian species Euflictis cyanophlyctis. Considering such studies, the LC50 from those species are lower than that observed in the present study to Laeonereis acuta (8.19 mg/L), except for T.

flumineus. These results demonstrated the higher resistance of L. acuta to this herbicide.

Such resilience may be related to the natural abiotic environmental fluctuations characteristic of estuarine regions that this species inhabits (Elliot and Quintino, 2007).

Previous literature has described a specific toxicity mechanism of Roundup as the interference in aerobic metabolism, the main energy generation pathway for aerobic organisms. In this sense, Peixoto (2005) demonstrated that Roundup exposure elicits a reduction in membrane potential as well as a 91% reduction in isolated mitochondrion ATP synthase activity of rat liver. These results provide support that this herbicide would interfere with enzymes involved in energy generation. Moreover, it has previously been demonstrated that, when the mollusks Bulinus truncatus were exposed to Herfosate (2.13 mg/L) for two weeks, lactate and glucose levels increased while glycogen and pyruvate levels declined (Bakry et al., 2015). Such effects suggest a possible metabolic switch towards anaerobic dependence, due to a reduction in cell respiration caused by herbicide exposure. Furthermore, exposure to 10 mg/L of Glyphosate II (Atanor) leads to an increase in AMP levels (99%), and causes a reduction in the adenylates energy charge in liver and muscle from the fish Odontesthes bonariensis (Menéndez-Helman et al., 2015). These studies demonstrate that glyphosate based herbicides would cause perturbations to the energetic balance within animal tissues. As a result, such alterations would induce an overall reduction to the oxygen utilization by tissues. However, no observation in the present study indicated an alteration in the global oxygen consumption in the polychaete L. acuta. Such an alteration would occur in a particular tissue or animal body region that may have been

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33 masked by an energetic imbalance in other parts of the animal. Regardless of no result being observed here regarding energetic balance differences, it remains important to consider ROS generation.

As ROS generation rates are directly linked with oxygen consumption (Storey, 1996), a possible alteration in oxidative metabolism would lead to differences in ROS levels. In the present study ROS levels in the posterior region from L. acuta were reduced following herbicide exposure. This observation may be related to the lower cuticle width; almost two times lower than other body regions (Rosa et al., 2005). Such reduction suggests that this body region, that possesses a lower diffusion barrier to permit gas exchange, is also more susceptible to the introduction and damaging effects of xenobiotic compounds.

It has been suggested that posterior region maintains a higher antioxidant capacity in comparison to other body regions due to its importance regarding contaminant uptake. However, even with a higher capacity to mitigate ROS, higher production of pro-oxidant molecules was demonstrated in this species (Ferreira-Cravo et al., 2007). In the present study, a reduction in ACAP levels following exposure to the higher herbicide concentration for 24h was observed in all body regions; however, these levels returned to normal after 96h. ACAP levels can be influenced by several factors, such as alterations in the components of the enzymatic and non-enzymatic antioxidant defense system (Amado et al., 2009). In this sense, studies indicate that exposure to glyphosate as well as it commercial formulation may cause alteration in some components of the antioxidant defense system. Contardo-Jara et al. (2009) verify that exposure for 96h to glyphosate or its formulations in concentrations ranging from 0.05 to 5 mg/L cause an increase in SOD, CAT and GST activity in the annelid Lumbricus variegatus. Alterations to the activity of several enzymes involved in the antioxidant

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34 defense system such as SOD, CAT, GST and GPx in the fish species Carassius auratus (Lushchak et al., 2009) and Prochilodus lineatus (Modesto e Martinez, 2010b), and SOD and CAT in the amazonian teleost surubim (Pseudoplatystoma sp.) exposed to Roundup (Sinhorin et al., 2014) have been demonstrated previously. These results are different from those observed in the present study, which indicate that the antioxidant enzymes and GSH from L. acuta were not altered after Roundup exposure. The antioxidant defense system is composed of both enzymes and low molecular weight ROS scavengers. Ascorbic acid, glutathione and other molecules are among ROS scavengers which represent important cellular defenses. Abele-Oeschger et al. (1994) demonstrated an augmentation to biliverdin levels in the Polychaeta Nereis diversicolor exposed to a pro-oxidant situation, an alteration considered the main antioxidant response. Biliverdin and bilirubin are formed by hemoglobin metabolism, with reactions catalyzed by hemeoxygenase enzymes in aerobic conditions; both molecules are considered important antioxidant molecules for polychaete species. Therefore, possible alterations in non-enzymatic antioxidants such as biliverdin, would be related to the reduction in ACAP levels in the posterior region from L. acuta exposed to Roundup.

However, in the present study, such molecules were not individually evaluated.

Moreover, a reduction in ROS levels would cause an alteration in oxidative balance and consequently cause alterations related to ACAP.

Alterations in oxidative balance such as ROS generation and ACAP levels would cause oxidative damage. Modesto and Martinez (2010a) and Sinhorin et al.

(2014) verified oxidative modifications in lipids and proteins in fish after glyphosate- based herbicide exposure. These authors suggest that damage induction would be related with ROS alterations as well as changes in antioxidant enzymes. Glusczak et al.

(2007) verify variation in lipid peroxidation in the fish Rhamdia quelen exposed to

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35 Roundup. These authors described a tissue specific pattern with induction in muscle, reduction in the brain, and maintenance of normal levels in liver. The present study observed a reduction in lipid peroxidation in all body regions from L. acuta after 24h of Roundup exposure, which was maintained after exposure to 96h in the posterior region.

However, an induction was observed in the middle region. Such a reduction would be directly related to a corresponding reduction in ROS production of tissues, as previously discussed. Although an alteration in ROS production and ACAP levels was caused by Roundup exposure, the absence of lipid damage demonstrates that L. acuta exposure to the highest Roundup concentration did not cause an oxidative stress situation. It is important to note that this animal species inhabits an estuarine region and this environment is naturally stressful for organisms due to oscillations in the physic- chemical parameters such as oxygen availability, pH and temperature (D’Incao et al., 1992; Elliott and Quintino, 2007). Such circadian variations demand that an animal possess an efficient antioxidant defense system, such as the maintenance of higher levels on its antioxidant components in order to neutralize such variations in metabolism. This stronger antioxidant defense system may promote resilience to the possible toxic effects of contaminants like Roundup. However, it has been demonstrated that Roundup possesses other toxicity mechanisms, such as impairment of the nervous system.

Cholinesterases are enzymes that possess important functions in the nervous system. In the present study the activity of acetylcholinesterase (AChE) and propionylcholinesterase (PChE) was analyzed. Roundup exposure for 96h caused a reduction in the activity of both enzymes present in the membrane fraction, although no difference was observed in the soluble fraction. In this context, glyphosate is an organophosphate pesticide (organophosphonate), which is classically described as

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36 different from the others by its incapacity to inhibit cholinesterase (Amarante Júnior et al., 2002). However, it was demonstrated that glyphosate and its commercial formulations would affect cholinesterase activity in vivo in fish species (Modesto e Martinez, 2010a) as well as in vitro (Sandrini et al., 2013; Braz-Mota et al., 2015).

Information regarding glyphosate inhibitory mechanism in ChE is scarce. However, two factors in the present study may contribute to such an effect: possible alterations in the production and liberation of ChE, and/or direct interaction of ChE with xenobiotics, resulting in conformational alteration and impairment in its activity. Sandrini et al.

(2013) provide further evidence for this in the bivalve mollusk Perna perna and in the fish species Danio rerio and Jenynsia multidentata wherein in vitro glyphosate exposure of tissue extracts cause direct inhibition in acetylcholinesterase activity.

Few studies have investigated the activity of cholinesterase isoforms such as propionylcholinesterase and butirylcholinesterase in animals under environmental stress. PChE is a pseudocholinesterase, representing an ancestral isoform which can break acetylcholine and other choline esters. Angelini et al. (2003) demonstrated the importance of PChE in acetylcholine breakdown in the sea urchin Paracentrotus lividus. Although highly important, there is no available information about glyphosate or Roundup in pseudochlinesterases; the present is study the first to demonstrate such effect.

It is important to note that herbicide effects in both AChE and PChE were observed only in the membrane fraction. This observation may be related with the intracellular environment in which the enzyme acts (membrane or soluble form). These cellular characteristics and enzyme cross-linking to cell structures would cause slight alterations in enzyme conformation and consequently alter its affinity to substrates or inhibitors. This effect was demonstrated in enzymes from other metabolic pathways,

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37 where the linkage to membrane phospholipids alters kinetic parameters to glycolitic enzymes (Pour-Rahimi and Nemat-Gorgan, 1987; Michaelidis and Beis, 1990).

5. Conclusion:

Roundup exposure causes toxicity to the Polychaeta Laeonereis acuta, specifically causing alteration in both ROS and ACAP levels, the latter of which is likely related to non-enzymatic antioxidants. It was further observed that Roundup exposure causes a perturbation in the cholinergic system via the reduction in AChE and PChE in the membrane fraction. Such biochemical alteration leads L. acuta to be more susceptible to other pro-oxidant agents present in the environment.

6. Acknowledgements

Fábio de Melo Tarouco, Robson Rabelo Velasques and Amanda da Silveira Guerreiro are graduate students fellow from the Brazilian agency CAPES - Brazil (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior). Filipe Guilherme Andrade de Godoi is an undergraduate student fellowed by the Scientific Initiation Program from CNPq (PIBIC - Conselho Nacional de Desenvolvimento Científico e Tecnológico).

Marcio Alberto Geihs is a Post-Doc fellow from CAPES. The authors would like to thank the financial support from CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico - process 480919/2013-5) and the National Institute of Science and Technology – Aquatic Toxicology (INCT-TA/CNPq).

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